The human olfactomedin 4 gene (OLFM4) encodes an olfactomedin-related glycoprotein. OLFM4 is normally expressed in a limited number of tissues, including the prostate, but its biological functions in prostate are largely unknown. In this study, we found that OLFM4 messenger RNA was reduced or undetectable in prostate cancer tissues and prostate cancer cell lines. To study the effects of OLFM4 on prostate cancer progression, we transfected PC-3 prostate cancer cells with OLFM4 to establish OLFM4-expressing PC-3 cell clones. The OLFM4-expressing PC-3 cell clones were found to have decreased proliferation and invasiveness compared with vector-transfected control PC-3 cells in vitro. In addition, nude mice injected with OLFM4-expressing PC-3 cells demonstrated reduced tumor growth and bone invasion and metastasis compared with mice injected with vector-transfected control cells. Mechanistic studies revealed that OLFM4 may exhibit its anticancer effects through regulating cell autophagy by targeting cathepsin D, as OLFM4 reduced cathepsin D protein levels and enzymatic activity and attenuated cathepsin D-induced cancer cell proliferation. In addition, overexpression of OLFM4 abrogated stromal cell derived factor-1 (SDF-1)-induced PC-3 cell invasiveness in a Matrigel invasion assay, partially through blocking SDF-1-mediated AKT phosphorylation. Coimmunoprecipitation and immunofluorescence staining studies in OLFM4-expressing PC-3 cells demonstrated a direct interaction between OLFM4 and cathepsin D or SDF-1. Taken together, these results suggest that OLFM4 negatively interacts with cathepsin D and SDF-1 and inhibits prostate cancer growth and bone metastasis. To further investigate the physiological functions of OLFM4 gene in the prostate tissue, we have developed and analyzed an OLFM4 knock out mouse model.During this last year, we found that OLFM4 loss is associated with prostate cancer progression in clinical samples and in a mouse model. In patient specimens, we found loss of heterozygosity at rs 2298231(S118S) within exon 2 of OLFM4 in cancerous prostate epithelial cells using laser-capture microdissection. We further found hemizygous and homozygous deletions of the OLFM4 gene in prostate cancer samples via fluorescence in situ hybridization analysis. Interestingly, OLFM4 knockout mice developed prostatic intraepithelial neoplasia (44%) and prostatic adenocarcinoma (28%) at ages 1824 months, and expression microarray analysis demonstrated increased levels of genes associated with cell proliferation in these prostate samples. In particular, loss of OLFM4 produced increased expression of the ETS family gene Ets1 and the cancer-promoting matrix metalloproteinase 19 (MMP19). In our most recent studies, we show that Olfm4-knockout mice sporadically developed prostatic intraepithelial neoplasia (44%), prostatic adenocarcinoma (28%), and other tumors at 1824 months of age. The gene-expression profile of prostate tissues from Olfm4-deficient mice revealed significant changes for genes associated with neoplastic progression. Human OLFM4 protein inhibited prostate stem/progenitor cell proliferation and mediated hedgehog signaling-pathway activities through direct interaction with sonic hedgehog protein. Taken together, our data indicate that OLFM4 plays a tumor-suppressor role in prostate neoplastic progression.